Plant Molecular Biology 38: 531–538, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Cloning and characterization of cold-regulated glycine-rich RNA-binding protein genes from leafy spurge (Euphorbia esula L.) and comparison to heterologous genomic clones David P. Horvath and Prudence A. Olson U.S. Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory, State University Station, Fargo, ND 58105-5674, USA Received 16 December 1997; accepted in revised form 10 April 1998

Key words: cold acclamation, gene regulation, glycine-rich RNA-binding protein

Abstract Leafy spurge (Euphorbia esula) is a perennial weed which is capable of acclimating to sub-freezing temperatures. We have used the differential display technique to identify and clone a cDNA for a cold-regulated gene (cor20) which hybridizes to mRNAs that accumulate specifically during the cold acclamation process. The cor20 cDNA was used to isolate two different genomic clones. Both clones were similar but not identical to each other and the cDNA. Sequence analysis of the genomic clones indicated that they share considerable homology to a group of glycine-rich RNA-binding protein genes. Comparison of the promoter region from the three clones (Ccr1 from Arabidopsis, BnGRP1O from Brassica napus, and GRRBP2 from Euphorbia esula) have identified at least two conserved motifs. CAGC is most likely involved in cold regulation and AACCCYAGTTA, is conserved but has no known function. RNAs which hybridize to cor20 reach maximal expression in less than 2 days after exposure of the plant to temperatures of 5 ◦ C, and remains at high levels in the plant for at least 30 days so long as the plant is left in the cold. These RNAs drop to control levels within 24 h when the plant is returned to normal growing temperatures. Transcripts which hybridize to cor20 do not accumulate under conditions of drought or heat stress. These transcripts are induced in response to low temperatures in roots, stems and leaves, but are expressed constitutively in tissue culture at control temperatures.

Introduction Euphorbia esula, commonly known as leafy spurge, is a noxious perennial weed that infests 2.5 million acres of land across the northern United States and Canada [15]. A primary feature of this plant is the existence of numerous axillary buds on its stem, crown and roots. Crown and root buds are capable of cold hardening Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AF03639 (GRRBP1) and AF031933 (GRRBP2).

and provide the plant with active meristems after winter kill of the above-ground portions of the plant [5]. These subterranean buds are thus responsible for the perennial nature of leafy spurge. Consequently, understanding the mechanisms that control cold hardening in root and crown buds could aid in developing measures for controlling leafy spurge and other perennial weeds with similar survival mechanisms. The biochemical changes correlated with cold acclimation have been extensively studied [13, 20, 22]. Increases in solutes such as carbohydrates and certain amino acids, changes in the lipid composition of the cellular membranes and changes in gene expression have been shown to occur in all plants that have been studied [13, 20, 22]. However, little is known about the mechanisms by which plants sense low temper-

532 ature, or the subsequent signals involved in altering the plant’s biochemistry. Recent success in cloning cold-induced genes from members of the Brassicaceae family (notably Arabidopsis thaliana and Brassica napus) have provided the first tools with which to study the signal transduction pathways that may bring about altered gene expression in response to cold [2, 7, 12, 16, 17, 18, 25]. Initial analysis of these genes have indicated that they are controlled primarily at the level of transcription initiation, and that specific enhancers are located in the promoters of several such genes (designated C-repeat or DRE for drought-responsive element) [10, 26]. The functionality of C-repeat/DRE appears to be conserved even in plants such as tobacco that lack the ability to acclimate to freezing temperatures [28]. In addition to the ability to confer cold regulation to a basal promoter, the C-repeat/DRE confers drought responsiveness as well [29]. This is not surprising since freezing stress brings about a dramatic decrease in a plant’s osmotic potential [6]. However, not all cold-regulated genes are also responsive to drought stress [2, 27]. Recent cloning of a factor CBF1, may help determine why some genes which contain C-repeat/DRE are responsive to drought and cold, or only cold [23]. In this paper, we describe the cloning by differential display of an individual member of the glycinerich RNA-binding gene family which hybridizes to cold regulated but not drought regulated RNAs, and subsequent isolation of two different homologues of this cDNA from a genomic library of leafy spurge. Expression patterns of these genes are identified and analysis of the promoter sequences for one of the genomic clones is presented.

chamber at 5 ◦ C under an 8 h photoperiod prior to harvest. Harvested plant material was frozen immediately in liquid nitrogen and stored at −80 ◦ C.

Materials and methods

Differential display and cloning

Plant material

The differential display was performed using a kit developed by Gene Hunter Corp. (Brooklyn, MA) as directed by the manufacturer. Reamplification of probes was done according to the manufacturer’s protocol, except that Tfl (Epicenter Technologies, Madison, WI) was used as the thermostable DNA polymerase. Twenty times Tfl buffer and MgCl2 (to a final concentration of 3.75 mM) was substituted for the Taq buffer supplied with the differential display kit. After reamplification, the resulting bands were blunt-ended by addition of 5 units of the large (Klenow) fragment of Escherichia coli DNA polymerase I directly to the 40 µl amplification reaction followed by a 30 min incubation at 37 ◦ C. Bands were gel-purified and used

Plants used for these experiments were started from shoot cuttings as a small group of plants originally isolated from a wild E. esula L. population in North Dakota. Shoot cuttings were placed in Sunshine I potting mix (Sun Gro Horticulture Inc., Bellevue, WA) and grown in a greenhouse under an 18 h photoperiod at 28 ◦ C. Plants were selected for experimentation when they were 2–3 months old. Tissue cultured plant material was started from hypocotyl sections and maintained on Gamborg’s B5 media (Gibco BRL, Grand Island, NY) supplemented with 0.1 mg/l 2,4D. For cold treatments, plants were placed in a growth

RNA isolation RNA was extracted using a modification of the method described by Schultz et al. [21]. Spun columns were prepared by mixing 2 g of insoluble polyvinylpyrolidone with 20 ml of RNA extraction buffer (10 mM Tris pH 7, 10 mM EGTA, 250 mM NaCl, 0.6% SDS, 343 mM p-aminosalicylic acid, and 28 mM triisopropyl-naphthalenesulfonic acid sodium salt). This mixture was loaded into 3 ml syringes plugged with glass wool. The resulting column was centrifuged at 1500 × g for 5 min to remove the excess buffer. One to two hundred mg of frozen plant material was ground to a fine powder in liquid nitrogen using a mortar and pestle. RNA extraction buffer (0.75 ml) was immediately added to the frozen powder, and grinding was continued until the homogenate thawed. The homogenate was then layered on top of the column. An additional 0.75 ml of RNA extraction buffer was used to wash the mortar and pestle. The resulting wash was added to the column. The column was again centrifuged at 1500 × g for 5 min. The eluate was extracted twice with an equal volume of phenol and three times with an equal volume of chloroform. The nucleic acids were ethanol-precipitated [19], and the resulting pellet was air-dried and resuspended in 100 µl of DEPC treated water. RNA was precipitated twice with lithium chloride, once with ethanol and air-dried [19]. The RNA was resuspended in a final volume of 100 µl of DEPC-treated water and quantified using standard spectrophotometric means [19].

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Figure 1. Northern blot analysis of cor20 gene transcripts from various plant tissues. Total RNA (10 µg) isolated from leafy spurge roots (R), stems (S), leaves (L) or disorganized callus culture (C) was separated on formaldehyde agarose gel, transferred onto nylon membrane and hybridized with 32 P-Iabeled cDNA of cor20 or a control message which was not cold-regulated. Temperatures indicate the treatment for 11 days prior to harvest.

as probes. Bands hybridizing to cold-regulated RNAs were ligated into the SmaI site of Bluescript for storage and sequencing. A genomic library of leafy spurge was prepared by Stratagene in the ZAP Express lambda phage vector. The cold-regulated cDNA was used as a probe to identify clones containing homologous sequences. Plasmids from these clones were rescued according to the manufacturer’s protocol, and restriction sites were determined. Pertinent fragments of the genomic clones were subcloned into Bluescript.

Figure 2. Southern blot analysis of leafy spurge genomic DNA. Twenty µg of DNA was digested with BamHI (B), EcoRI (E), HindIII (H), PstI (P), SmaI (S), or XbaI (X), separated on a 1% agarose gel, blotted onto nylon membrane and hybridized with 32 P-labeled cDNA of cor20.

Sequencing Plasmid DNA was isolated from E. coli using a boiling mini-prep method [19]. Double-stranded sequencing was carried out by the DNA sequencing facility at Iowa State University. Southern and northern hybridizations

Figure 3. Northern blot analysis of cor20 gene transcripts after 24 h heat stress. Temperatures indicate the treatment prior to harvest. Total RNA (10 µg) isolated from leafy spurge leaves was separated on formaldehyde agarose gel, transferred onto nylon membrane, and hybridized with 32 P-labeled cDNA of cor20 or a control message which was not cold-regulated.

Southern and northern blotting and hybridizations were done according to Sambrook et al. [19], except that all final washes were done in 2× SSC at 65 ◦ C. RNase protection assays To obtain the probe for the RNase protection assays, the 172 bp HindIII/BamHI fragment from the 50 end of GRRBP1 was subcloned into Bluescript II SK+. The sub-clone was cut with BamHI and a radioactive RNA probe was made by transcribing the plasmid using T7 RNA polymerase with the MAXIscript In Vitro Transcription Kit (Ambion, Austin TX). As an additional size standard, SmaI-cut Bluescript was also transcribed using T7 RNA polymerase to give a 73 nt fragment. The probe was gel purified and used in the

Figure 4. Northern blot analysis of cor20 gene transcripts after drought stress. Plants were drought stressed (D) by withholding water until the leaves were visibly wilted or treated at the indicated temperatures. Total RNA (10 µg) isolated from leafy spurge leaves was separated on formaldehyde agarose gel, transferred onto nylon membrane, and hybridized with 32 P-labeled cDNA of cor20 or a control message which was not cold-regulated.

534 grown cells, it was found that cor20 hybridized to RNAs that accumulated to high levels in both the control and cold-treated callus. Southern blot analysis of cor20 Figure 5. Time course of cor20 gene transcript accumulation or loss during acclamation and deacclimation. Total RNA (10 µg) isolated from leafy spurge tissue cultured cells after various times (in days) at or after (days deacclimated) 5 ◦ C, was separated on formaldehyde agarose gel, transferred onto nylon membrane, and hybridized with 32 P-Iabeled cDNA of cor20 or a control message which was not cold-regulated.

RNase protection assays according to guidelines contained in the RPA II Ribonuclease Protection Assay Kit (Ambion). The resulting fragments were separated on an 8% denaturing polyacrylamide (sequencing) gel.

Southern blot analysis of the leafy spurge DNA was used to obtain an indication of the genomic organization and copy number of the cor20 gene. Leafy spurge DNA was digested with six different restriction endonucleases (BamHI, EcoRI, HindIII, PstI, SmaI, and XbaI), separated on a 1% agarose gel, blotted and probed with the cor20 clone (Figure 2). Cor20 hybridized strongly to several bands and weakly to a number of other bands in all of the digests tested indicating that cor20 most likely represents one member of a gene family.

Results

Cor20 homologues are not induced by drought or heat stress

Cloning of a cold-regulated gene by differential display and tissue specificity of expression Earlier studies with leafy spurge indicated several biochemical changes in root material of leafy spurge upon cold treatment (Frear and Swanson, unpublished results). RNA was isolated from three separate sets of leafy spurge roots before and after the plants were subjected to 22–34 days at 5 ◦ C. These RNA samples were then used in a differential display experiment to identify mRNAs that accumulated during the cold treatment. Nine mRNAs were identified and the corresponding cDNA for each message was amplified by PCR and used to probe northern blots. One of the cDNAs (cor20) consistently accumulated in cold-treated plants. It was determined that cor20 hybridizes to one or more RNAs ca. 1000 bp in size that accumulate in the cold-treated root. This cDNA was cloned for further studies. In order to determine if expression of genes homologous to cor20 was limited to specific plant tissues, control and cold-treated leaf, stem, root and disorganized callus cultures of leafy spurge grown on B5 medium supplemented with 0.1 mg/l 2,4-D were collected. RNA from these samples was isolated and subjected to northern blot analysis using the cor20 clone as a probe (Figure 1). Results from this experiment show that RNAs homologous to cor20 accumulate in roots, stems and leaves of the tissues tested in response to the cold treatment. Surprisingly, in tissue-culture

Northern blot hybridization experiments were done to determine if cor20 homologues were induced specifically by cold temperatures, or if other environmental stresses could also bring about the accumulation of this RNA. RNA was isolated from plants that had been subjected to heat stress (24 h at 40.5 ◦ C) and drought stress (water was withheld until the plants were visibly wilted) as well as control and cold-treated plants. The results indicate that cor20 homologues are not induced by heat or drought stress (Figures 3 and 4, respectively). Time course of cor20 accumulation and loss In order to determine the kinetics of RNA accumulation for cor20 homologues, leafy spurge plants were placed at 5 ◦ C, and samples of leaf material were collected at 0, 2, 6, 14, 21 and 30 days. In addition, plants were taken out of the cold after 30 days and moved to normal growing temperatures. Again, samples of root material were collected at 0, 1 and 3 days after removal from the cold. RNA from these samples was isolated and subjected to northern blot analysis and probed with the cor20 clone. Transcript tah hybridized to cor20 accumulated to maximal levels in under 2 days after cold treatment, and remained high throughout the experiment (Figure 5). Also, these RNAs returned to control levels within 1 day after the return of the plants to normal growing temperatures.

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Figure 6. Comparison of key promoter and coding sequences as well as intron location of Cor20, GRRBP1, GRRBP2, Ccr1 and BnGRP10. Conserved sequences in the promoter and the RNA binding domains are underlined. The TATA box motifs are in bold characters.

Cloning and sequence analysis of cor20 genomic clone The cor20 cDNA was used as a probe to identify two homologous genomic clones (GRRBP1 and GRRBP2, GenBank accession numbers AF036339 and AF031933 respectively). Subclones of the suspected coding region were obtained and sequenced. Analysis of the sequences indicated that both genes were similar but not identical to each other, and that neither was identical to the orginal cDNA (Figure 6). A search of the available gene banks using the BLASTP 1.4.9MP program (3/26/1996) indicated that both genomic clones had homology to a number of glycinerich RNA-binding proteins with the most similar being a gene from rice (smallest sum probability 1.6e-48). Both genomic clones appear to contain a single intron, and one of the clones (GRRBP2) contained more than 500 bp of promoter sequences. Only two other genomic clones of cold-regulated glycine-rich RNAbinding protein genes were found after an extensive search of the existing sequence databases (Ccr1 from A. thaliana and BnRG10 from B. napus). The position of the observed intron is conserved to the base pair in both genomic clones from leafy spurge and the heterologous genomic clones (Figure 6). A comparison of the promoter sequence of GRRBP2 with two other cold-regulated glycine-rich RNA-binding protein genes which were not regulated by drought

(BnRG10 from B. napus and Ccr1 from A. thaliana) indicated the presence of two conserved motifs. One was a CAGC element located between 46 and 73 bp 50 to the TATA box elements and the other is an AACCCYAGTTA located 23–28 bp 50 to the TATA box. RNase protection assays In order to ascertain which, if any of the two genomic clones were cold-regulated, and to look for cold-regulated expression of other individual members of this gene family, RNase protection assays were performed using the 50 end of the genomic clone GRRBP1 as a probe. The results of this experiment indicated that there were several major groups of RNAs (about 145, 134 and 98 nt in length) protected by the probe in cold-treated roots, stems leaves and callus as well as in callus grown under control temperatures which were differentially expressed. The 145 and 98 nt bands are within several nt of the expected size for protected fragments from GRRBP1 and GRRBP2 respectively. The 134 nt band most likely represents a different cold-regulated member of this gene family. There are 3 additional groups of bands (located between the 134 and 98 nt bands) which are expressed preferentially in callus. It is likely that groups of bands are the result of partial digestion of single RNAs, since the number of bands within given groups varied from gel to gel (data

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Figure 7. RNase protection assay. Total RNA (10 µg) from callus, roots, stems, leaves and yeast were hybridized to 32 P-labeled RNA probe from the 50 end of GRRBP1. Hybridized RNAs were digested and separated on a 8% denaturing polyacrylamide gel. Approximate size of fragments is given in nucleotides, and the temperature (22 ◦ C and 5 ◦ C) indicate growth conditions 11 days prior to harvest.

not shown). These results are consistent with the idea that both GRRBP1 and GRRBP2 are cold-regulated.

Discussion In the recent past, many genes with transcripts that accumulate during cold acclamation have been identified by differential hybridization [11]. Like most previously cloned cold-regulated genes, cor20 hybridizes to RNAs induced by low temperatures in all tissues examined, and the kinetics of cor20 expression is similar to that observed for many previously cloned cold-regulated genes [7]. The cloned cDNA was used to identify two genomic clones GRRBP1 and GRRBP2. The original cDNA clone is only ca. 85% identical to the genomic clones as indicated by sequence analysis. The differences between these two clones indicates that they are all likey to be representative of different members of the same gene family. Sequence analysis of other members of this gene family will be required to determine the level of conservation between members of

this gene family in leafy spurge. Homology searches of the sequence databases indicate that cor20 genomic clones had significant homology to a class of glycinerich RNA-binding protein genes that are responsive to a number of different environmental conditions and which recently have been identified in plants [1, 3, 8, 9, 14, 24]. The cor20 cDNA clone hybridizes strongly to as many as three bands and weakly to as many as 10 bands in Southern blot experiments with genomic DNA of leafy spurge. Thus, cor20 also appears to be a member of a gene family as is the case in other RNA-binding proteins [2]. Only two other genomic clones of cold-regulated glycine-rich RNA-binding protein genes have published sequence. One is Ccr1 (GenBank accession number L04171) from A. thaliana which is induced not only by cold, but is also regulated by circadian rhythm [2], and the other is BnGRP10 GenBank accession number Z14143) from B. napus [1]. Unlike many of the coldregulated genes that have been described, these genes are not induced by drought stress [1, 2]. Comparisons between the promoters of Ccr1 (kindly provided by Dr Joel Kreps), BnGRP10 and GRRBP2 identified two conserved sequence motifs. One was a CAGC (CAGCC in BnGRP10 and cor20) located between 46 and 73 bp 50 to the suspected TATA box elements in all of the promoters. This sequence is identical to the core component of the C-repeat/DRE that has been identified in several other cold-regulated genes from plants [10, 26]. Interestingly, the sequences surrounding the core CGAC element in GRRBP1, Ccr1 and BnGRP10 are markedly different from those found in genes responsive to both cold and drought. It has been suggested that sequences surrounding the core CGAC element determine which member of the AP2 family of DNA binding factors interact with a given element [23], If there are different members of this family which are responsive to drought or cold or both, the differences in surrounding sequences could offer an explanation for why some genes with C-repeat/DRE are responsive to both drought and cold whereas others are only responsive to cold. However, it should be noted that there are no obvious patterns in the sequences surrounding the C-repeat/DRE in GRRBP1, Ccr1, Cor15b and BnGRP10, and thus it is also possible that other regulatory elements might be involved in this phenomenon. One other sequence that appears to be highly conserved is the AACCCYAGTTA element located between 23–28 bp 50 to the TATA box. The function of this element is unknown, however, the fact that it is conserved between both the Brassica

537 and the Euphorbia indicates that it most likely plays a significant role in the regulation of these genes. The Ccr transcripts are also regulated by circadian rhythm at both cold and control temperatures (albeit at much higher basal levels in the cold) (Kreps, unpublished results). Also, other members of the same gene family from other species (Nicotinia tabacum, Zea mays, and Daucus carota) are induced by other environmental stresses such as drought and wounding, but not by cold [3, 9, 24]. It is not yet known if any cor20 homologues are influenced by circadian rhythm, but both GRRBP1 and GRRBP2 and several other related genes are constitutively expressed at high levels in tissue culture. This observation is consistent with the possibility that at least some cor20 homologues are responsive to other environmental stimuli. It is also possible that the constitutive expression is in response to a nutrient, hormonal or developmental signal experienced by the cultured cells. Since all of the members of this class of genes appear to be responsive to stress, insight into the function of these genes might be gained by altering the culture conditions and observing changes in expression patterns and cell physiology.

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Acknowledgements 15.

The authors would like to thank Drs Larry Heilmann and Sarah Gilmour for critically reviewing the manuscript, Kathy Circle for technical help in cloning of the genomic constructs, and Dr Joel Kreps for providing us with sequences from the Ccr1 promoter.

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